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App ca1 paula

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HIPPOCAMPUS 00:000–000 (2010)RAPID COMMUNICATION Layer-Speciﬁc Alterations to CA1 Dendritic Spines in a Mouse Model of Alzheimer’s Disease P. Merino-Serrais,1 S. Knafo,1,2* L. Alonso-Nanclares,1 I. Fernaud-Espinosa,1 and J. DeFelipe1ABSTRACT: Why memory is a particular target for the pathological et al., 2009a, in press). Apart from amyloid plaques,changes in Alzheimer’s Disease (AD) has long been a fundamental ques- the Ab peptide accumulates in different forms in AD:tion when considering the mechanisms underlying this disease. It hasbeen established from numerous biochemical and morphological studies intracellular Ab and oligomeric Ab. Indeed, it hasthat AD is, at least initially, a consequence of synaptic malfunction been demonstrated that spine heads are targets ofprovoked by Amyloid b (Ab) peptide. APP/PS1 transgenic mice accumu- oligomeric Ab (Lacor et al., 2007), and it has beenlate Ab throughout the brain, and they have therefore been employed to suggested that targeting and functional disruption ofinvestigate the effects of Ab overproduction on brain circuitry and particular synapses by Ab oligomers may provide acognition. Previous studies show that Ab overproduction affects spinemorphology in the hippocampus and amygdala, both within and outside molecular basis for the speciﬁc memory loss in ADplaques (Knafo et al., (2009) Cereb Cortex 19:586-592; Knafo et al., (Lacor et al., 2007). Nevertheless, previous studies(in press) J Pathol). Hence, we conducted a detailed analysis of dendri- indicate that there is only a weak correlation betweentic spines located in the stratum oriens and stratum radiatum of the plaque load and cognitive functions (Terry et al.,CA1 hippocampal subﬁeld of APP/PS1 mice. Three-dimensional analysis 1991). In fact, plaques are sometimes detected even inof 18,313 individual dendritic spines revealed a substantial layer-speciﬁcdecrease in spine neck length and an increase in the frequency of spines nondemented patients (Price et al., 2009) and cogni-with a small head volume. Since dendritic spines bear most of the tive decline is better reﬂected by the level of solubleexcitatory synapses in the brain, changes in spine morphology may be Ab oligomers, distributed diffusely outside the plaquesone of the factors contributing to the cognitive impairments observed in (Selkoe, 2002). Moreover, at least in aged micethis AD model. V 2010 Wiley-Liss, Inc. C bearing AD mutations, plaques occupy a negligibleKEY WORDS: morphology; confocal microscopy; amyloid beta; fraction of the neuropil, less than 5% (Cohen et al.,unbiased stereology; amyloid precursore protein 2009;Knafo et al., 2009a, in press), and therefore, it is unlikely that cognitive impairment in these mice arises solely from changes in synapses within the plaques. Hence, AD neuropathological research is INTRODUCTION increasingly focusing on the changes in plaque-free regions of the neuropil and in AD-like pathology The presence of Ab plaques is one of the pathological hallmarks of prior to plaque appearance. Here we have analyzedAD, and they have been associated with changes in neurite morphology dendritic spines in the CA1 subﬁeld, an area criticaland dendritic spine density (Tsai et al., 2004; Spires et al., 2005; Knafo for spatial orientation and learning (Andersen et al., 2006), in order to determine the microstructural basis1 of the hippocampal-dependent cognitive impairment Instituto Cajal (CSIC), Madrid, Spain and Laboratorio de Circuitos Cor- ´ ´ ´ticales, Centro de Tecnologıa Biomedica, Universidad Politecnica de in APP/PS1 mice (Malm et al., 2007). DendriticMadrid, Madrid, Spain; 2 Centro de Biologıa Molecular ‘‘Severo Ochoa’’, ´ spines represent the major postsynaptic elements of ´Consejo Superior de Investigaciones Cientıﬁcas (CSIC)-Universidad excitatory synapses in the cerebral cortex (Gray, 1959) ´Autonoma de Madrid, Madrid, Spain and they are fundamental to memory, learning andAdditional Supporting Information may be found in the online version ofthis article. cognition (Lamprecht and LeDoux, 2004). DendriticP. Merino-Serrais and S. Knafo contributed equally to this work. spines undergo remarkable activity-dependent struc-Grant sponsor: CIBERNED; Grant number: CB06/05/0,066; Grant spon- tural changes (Lang et al., 2004; Tsai et al., 2004)sor: EU 6th Framework Program; Grant number: PROMEMORIA LSHM- and they are targets of oligomeric Ab (Lacor et al., ´CT-2,005–512012; Grant sponsor: Spanish Ministerio de Educacion Cien- 2007). Therefore, spine morphology may be associ- ´cia e Innovacion; Grant numbers: BFU2006–13395, SAF2009–09394, ´BES-2,007–16542; Grant sponsors: Fundacion CIEN (Financiacion de ´ ated with Ab pathology and synaptic malfunction. We ´Proyectos de Investigacion de Enfermedad de Alzheimer y enfermedades show that in APP/PS1 mice, CA1 spines necks are sig-relacionadas 2008), Ministry of Science and Technology. niﬁcantly shorter in the stratum oriens. In addition, ´*Correspondence to: Dr S. Knafo, Centro de Biologıa Molecular ‘‘Severo the frequency of spines with a small head augments in ´Ochoa’’, Consejo Superior de Investigaciones Cientıﬁcas (CSIC)-Universi- the same stratum radiatum of the same subﬁeld. These ´dad Autonoma de Madrid, Madrid, Spain. E-mail: sknafo@cbm.uam.esAccepted for publication 25 June 2010 ﬁndings indicate that circuits in the stratum radiatumDOI 10.1002/hipo.20861 and the stratum oriens might be affected differently byPublished online in Wiley Online Library (wileyonlinelibrary.com). AD-related mutations. In addition, this study suggestsV 2010C WILEY-LISS, INC.

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2 MERINO-SERRAIS ET AL.that dendritic spine morphology reﬂects the synaptic malfunc- tracing. To assess the morphology of spines, a solid surface thattion arising from Ab overexpression. exactly matched the contours of the head was constructed for We used a transgenic mouse line (12 to 14-month-old male each spine using Imaris (Fig. 1i), and the length of the spinemice) expressing a Mo/Hu APP695swe construct in conjunc- neck was measured manually in three-dimensions using thetion with the exon 9 deleted variant of human presenilin 1 same software (Isosurface module, see detailed methods). To(PS1-dE9: (Scheuner et al., 1996). Age-matched littermates estimate the density of plaques, plaques were immunostainedserved as controls (Tg-). The mice were perfused with 4% with an anti-Ab antibody in serial sections taken from theparaformaldehyde and coronal sections of the ﬁxed brain were same mice, and unbiased stereology rules were applied usingobtained. A total of 270 pyramidal neurons from Tg2 mice optical fractionation and the Nucleator probe (Moller et al.,and 262 neurons from APP/PS1 mice were microinjected 1990). For all the morphological parameters measured, the val-individually with Alexa594 (Invitrogen, Eugene, OR, ues were averaged to give a neuron mean, and neurons fromFigs. 1a,b), and plaques were counterstained with thioﬂavin-s each animal were averaged for the animal mean. Normality wasafter injection (Figs. 1a–c). The plaques and dendrites in the tested using the Kolmogorov-Smirnov test and a two-tailedstratum oriens (corresponding to basal dendrites) and in the unpaired t-test was used to test for the overall effect. Whenstratum radiatum (collateral apical dendrites) were scanned with more than two groups were compared, a one-way ANOVA wasa Leica laser scanning multispectral confocal microscope (TCS used, followed by Tukey’s Multiple Comparison post hoc test.SP5) using 488 and 594 nm laser lines. Image stacks (Physical Data are presented as the mean 6 SEM.size 76.9 3 76.9 lm, logical size 1,024 3 1,024 pixels) con- We examined 1,475 amyloid plaques and 532 injectedsisted of 100–350 image planes. A 633 Glycerol-immersion pyramidal neurons by confocal microscopy (Fig. 1a). Welens (NA, 1.3; working distance, 280 lm; refraction index, encountered only ﬁve dendrites that passed within plaques in1.45) was used with a calculated optimal zoom factor of 3.2 the stratum oriens (basal dendrites) and no such dendrites inand a z-step of 0.14 lm (voxel size, 75.1 3 75.1 3 136.4 the stratum radiatum (apical dendrites). Typical plaques thatnm). These settings and optics represent the highest resolution were positive for thioﬂavin-s consisted of a core surrounded bycurrently possible with confocal microscopy. After acquisition, a diffuse less dense ring. The dendrites passing through the pla-the stacks were processed over 10 iterations with a three-dimen- ques were located in the diffuse peripheral ring, as describedsional blind deconvolution algorithm (Autodeblur; Autoquant, previously (Cruz et al., 1997; Knafo et al., 2009a, in press).Media Cybernetics) to reduce the out-of-focus light, and Dendrites were categorized according to their location withthereby removing the haze and the blur, restoring vital details respect to the Ab plaques, as: (1) dendrites from transgene-to the datasets (Supporting Information Fig. 1b). The stacks negative (control) mice (Tg-); (2) dendrites located in awere then opened with Imaris 6.0 (Bitplane AG, Zurich, Swit- plaque-free area (Plaque-free); (3) segments of dendrites withinzerland), a three-dimensional image processing software. In a plaque (Plaque).stacks containing images of Ab plaques (green), the green chan-nel was deleted and the stacks were coded (the codes were notbroken until the quantitative analysis had been completed). Spines Have a Shorter Neck in Plaque-FreeSpine density measurements and their morphology were Regions of the Stratum Oriens of APP/PS1 Miceassessed by another investigator using only the red channel to The spine density in the stratum oriens was signiﬁcantlyassure impartiality (Figs. 1f,i). For spine density measurements, different among the three categories of dendrites (P 5 0.006,image stacks were viewed with a computerized data collection one-way ANOVA, Fig. 1d). Accordingly, the spine density wassystem (Neurolucida 7.1 Confocal module; MicroBrightﬁeld, signiﬁcantly lower within plaques than in other categories ofInc., Williston, VT), the image of the acquired dendrites was dendrites (0.85 6 0.17 spines/lm, N 5 4). However, spinetraced in three-dimensions and the spines were marked during density for Plaque-free dendrites (1.33 6 0.054, N 5 7) didFIGURE 1. Spines are shorter in the stratum oriens of APP/ in Tg- and APP/PS1 mice in plaque-free regions. Note the similar-PS1 mice. (a) A panoramic view of neurons injected with Alexa ity in spine density along the length of the dendrite. (f ) Represen-594 (red) and thioﬂavin-s positive plaques (green) in the hippo- tative projection images of dendrites from Tg- and APP/PS1 micecampus (203, oil). (b) Representative projection images (403, oil) (633, glycerol). Necks are marked as was done for measurementsof injected neurons and plaques in Tg- (left) and APP/PS1 mice (g) Decreased average neck length for spines in APP/PS1 mice(right). The plaques seen in the stratum radiatum (apical outside plaques. (h) Cumulative frequency plots showing the dis-dendrites) are located close to labeled dendrites but they do not tribution of spine neck length, indicating a shift towards lowercontain dendrites, as determined by three-dimensional analysis. values in the entire spine population. (i) Representative images(c) An example of a dendrite located in the stratum oriens passing of dendritic segments with the contours of the spine heads ofthrough a plaque and showing a decrease in spine density within APP/PS1 mice constructed for each spine (see Supporting Infor-the plaque. (d) Spine density is decreased signiﬁcantly within the mation Fig. 1 for details). (j-k) The head volume is similar for Tg-plaques. Note that the spine density is similar in Tg- mice and in and APP/PS1 mice. *P < 0.05, Tukey’s Multiple Comparison postthe plaque-free areas of APP/PS1 mice. (e) The spine density as a hoc test. Scale bar, (a) 250 lm, (b) 25 lm, (c) 5 lm (f ) 0.6 lm (i)function of the distance from the soma (Sholl Analysis) is similar 0.8 lm.Hippocampus

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4 MERINO-SERRAIS ET AL.FIGURE 2. Increased frequency of spines with a small head in spine neck length, indicating a similar distribution for both spinethe stratum radiatum of APP/PS1 mice. (a) Representative projection populations. (f) The frequency of small spines increases substantiallyimages of dendrites from Tg- and APP/PS1 mice (633, glycerol). (b) in plaque-free areas of APP/PS1 mice. (g) Cumulative frequencyThe spine density is similar in Tg- mice and in the plaque-free areas curves showing a shift towards smaller head volumes in APP/PS1of APP/PS1 mice. (c) Spine density as a function of distance from mice. (h) A bar graph depicting a signiﬁcant increase in the frequencyapical trunk is similar in Tg- mice and APP/PS1 mice in plaque-free of small spines (volume < 0.03 lm3) in the stratum radiatum ofregions. (d) Constant average neck length for spines in APP/PS1 APP/PS1 mice. Scale bar, 0.6 lm.mice. (e) Cumulative frequency plots showing the distribution ofnot differ signiﬁcantly from that of control (Tg-) dendrites among the two categories of dendrites (P 5 0.88, t-test,(1.348 6 0.070, N 5 6). Moreover, a Sholl analysis of the Figs. J,k), and was 0.056 6 0.003 lm3, for Tg- dendrites (N 5spine density at different distances from the soma revealed that 6, 5,709 spines) and 0.055 6 0.003 lm3 for plaque-freethe spine density for Plaque-free dendrites was similar to that dendrites (N 5 7, 5,145 spines). Thus, in the stratum oriensof control dendrites over their entire length (Fig. 1e). of APP/PS1 mice, spine density is decreased within plaques and Spine neck length and head volume were measured in three spines are shorter outside plaques.dimensions in confocal image stacks. Because of the extensiveloss of spines within plaques, the spines within plaques (31 Small-Headed Spines Are More Frequentspines) were not included in the analysis of head and neck in Plaque-Free Regions of the Stratumdimensions. Signiﬁcant differences in the average spine neck Radiatum of APP/PS1 Micelength were found between the two dendritic categories (P 50.018, t-test, Figs. 1g,h). Spines in the APP/PS1 mice had a Spine density and morphology were examined in apicalsigniﬁcantly shorter neck (26%) than spines in Tg- mice spines branches protruding from the main apical trunk. These den-(0.548 6 0.040). Head volume was not signiﬁcantly different drites were located up to 300 lm from the stratum pyramidaleHippocampus

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ALTERED SPINES IN APP/PS1 MICE 5(cell body layer). Dendrites located in the stratum lacunosum- TABLE 1.moleculare were not included in the analysis. The average spinedensity along the apical dendrites in the stratum radiatum did Plaque Volume and Densitynot differ signiﬁcantly in APP/PS1 mice when compared with S. Oriens S. Radiatumthe control mice (1.71 6 0.08 for Tg- mice and 1.83 6 0.08 Parameter (N 5 4) (N 5 5)for APP/PS1 mice: P 5 0.62, student t-test; Figs. 2a,b). ASholl analysis revealed that the density of spines on Plaque-free Estimated total number of plaques 1551 6 272.6 1985 6 183.8dendrites was not signiﬁcantly different to that of control (Tg-) Plaque density (plaque/mm3) 1138 6 214.4 1174 6 113.0dendrites over their entire length (Fig. 2c). By contrast to Individual plaque volume (lm3) 0.019 6 0.002 0.029 6 0.003the stratum oriens, the neck length was similar in both groups Volume occupied by plaques (%) 1.421 6 0.201 1.554 6 0.195(P 5 0.43, t-test, Figs. 2d,e), as it was 0.519 6 0.017 lm forTg- dendrites and 0.494 6 0.024 lm in APP/PS1 mice. Theaverage head volume in this layer did not differ signiﬁcantly whereas the neck was signiﬁcantly shorter in the stratum ori-between Tg- dendrites (0.038 6 0.003 lm3: N 5 6; 4,437 ens of APP/PS1 mice. Moreover, spines in the stratum oriensspines) and plaque-free regions (0.033 6 0.003 lm3: N 5 7; are affected differently to spines in stratum radiatum, even6,044 spines. P 5 0.29, t-test, Fig. 2f ). Nevertheless, the within the CA1. These layer-speciﬁc morphological altera-cumulative frequency curves clearly indicated a distinct distri- tions are underscored by the fact that Ab plaques occupy abution of head volumes in the smaller values (Fig. 2g) and similar total volume of both layers (Table 1), implying a sim-indeed, a signiﬁcant increase (29%, P 5 0.01, t-test) in the ilar Ab load. Thus, we conclude that AD-related mutationsfrequency of small-headed spines (head volume < 0.03 lm3) have distinct effects on spines depending on their location.was evident in the plaque-free areas of APP/PS1 mice We also show here that dendrites within plaques are deﬁcient(Fig. 2h). Thus, we found an increase in the frequency of in spines, in accordance with observations in other brainsmall-headed spines in the stratum radiatum of APP/PS1 mice. regions (Tsai et al., 2004; Knafo et al., 2009a, in press). Spine loss within Ab plaques can affect local synaptic cir- cuits. However, since plaques occupy a minor fraction of theAmyloid Plaques Occupy a Small CA1 (below 2%), the morphological changes observed out-Fraction of the CA1 side plaques are more likely to contribute to the synaptic and We have described changes in dendritic spines within and cognitive impairments found in APP/PS1 mice (Malm et al.,outside of plaques that can affect local synaptic circuits. To 2007). We also found that spine density outside plaques isquantitatively determine the impact of plaques on CA1 connec- unchanged in APP/PS1 mice, in accordance to our previoustivity, we immunocytochemically stained Ab plaques in serial studies into the dentate gyrus and amygdala (Knafo et al.,sections of APP/PS1 brains (Supporting Information Fig. 2). 2009a, in press). These ﬁndings imply that cognitive impair-Using unbiased stereology, we then determined the total ment in these mice (Malm et al., 2007) does not arise fromnumber of plaques and their volume in the stratum oriens and changes in spine density in plaque-free areas. Rather, it isstratum radiatum, from which we could calculate the total likely that changes in spine morphology outside plaques con-volume occupied by the plaques. The estimated total number tribute to these cognitive deﬁcits.of CA1 plaques per mouse in one hemisphere was 1,551 6 The data presented here shows that the average spine neck272.6 (range, 1,045–2,081 plaques/mouse; N 5 4: Table 1) in length is shorter in the stratum oriens of APP/PS1 mice, boththe stratum oriens and 1985 6 183.8 (range 1,533–2,628 within and outside of the plaques. The morphology of theplaques/mouse, N 5 5) in the stratum radiatum. The density spine neck fulﬁls a key role in controlling the time windowof plaques in the stratum oriens was 1,138 6 214.4 plaques/ compartmentalization of calcium and other second messengersmm3, while it was 1,174 6 113.0 plaques/mm3 in the stratum in spines (Yuste et al., 2000). The shortening of the spine neckradiatum (Table 1). The average plaque volume was 0.019 6 in APP/PS1 mice may increase the diffusion between the0.002 mm3 in the stratum oriens and 0.029 6 0.0,029 mm3 in spine and the dendrite. Spines with a fast diffusion equili-the stratum radiatum (P 5 0.04, t-test). The estimated volume bration along the spine neck may be unable to retain secondoccupied by Ab plaques was 1.421 6 0.201% in the stratum messengers or activate proteins upon the LTP inducing stimu-oriens and 1.554 6 0.195% in the stratum radiatum (Table 1). lus (Bloodgood and Sabatini, 2005). This altered plasticityThese results suggest that under our experimental conditions, Ab may eventually contribute to the cognitive impairment seen inplaques occupy a relatively small fraction of the CA1 neuropil. APP/PS1 mice (Malm et al., 2007). This study shows that dendritic spines in the CA1 subﬁeld are We also found that APP/PS1 mice have a higher proportionsigniﬁcantly affected by Ab, both within plaques and in plaque- of spines with a small head volume in the stratum radiatum.free regions. Importantly, some changes in spine morphology in Importantly, small spines are more abundant after processesthis region were not evident in the dentate gyrus (Knafo et al., of long-term depression (LTD), a form of synaptic plasticity2009a) or the amygdala (Knafo et al., 2009b) when studied signiﬁcantly enhanced in many models of AD (Shankar et al.,with the same tools. For example, no signiﬁcant differences 2008). Spine head size determines the size and duration ofwere found in the length of spine neck in these regions, synaptic Ca21 transients (Majewska et al., 2000) and it is Hippocampus

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6 MERINO-SERRAIS ET AL.therefore correlated with the magnitude of signals transmitted Spine densityto the dendritic shaft (Harris and Stevens, 1989; Murthy et al., Dendritic spine density was determined by tracing the2000). Spines with smaller heads have smaller postsynaptic image of the acquired dendrites in three dimensions (withdensities (Harris and Stevens, 1989) and contain less AMPA Neurolucida). Spines were marked during tracing and all pro-receptors on their heads when compared to spines with larger trusions were considered as spines, applying no correction fac-heads (Kharazia and Weinberg, 1999), resulting in less sensitiv- tors to the spine counts. After tracing all the dendrites, eachity to glutamate at these spines (Matsuzaki et al., 2001). It is 2-channel stack (containing the green channel with amyloidtherefore possible that the increase in the frequency of small- plaques) was viewed with Imaris and we determined whetherheaded spines reﬂects long-term synaptic depression, thereby a dendrite entered a plaque (Knafo et al., 2009a). The tracedcontributing to the cognitive impairment seen in this AD dendrites were viewed with Neurolucida and the correspond-model. Moreover, it is possible that the greater frequency of ing stack with the green channel was opened. The traced den-spines with large heads in this layer reﬂects the loss of LTP drites were categorized as dendrites that passed through anin APP/PS1 mice (Trinchese et al., 2004). Therefore, the amyloid plaque, or dendrites whose entire length was in a pla-laminar speciﬁc changes observed in spine length might have que-free area. The reconstructed data were exported to Neuro-an important functional consequence in certain hippocampal lucida Explorer (MicroBrightField Inc., Williston, VT) forcircuits. In summary, we show here that spines in the CA1 are quantitative analysis. Spine density was calculated for eachmorphologically modiﬁed, which may reﬂect the functional dendrite by dividing the dendritic length by the number ofalterations at synapses induced by Ab overexpression in APP/ spines. Spine density was also analyzed as a function of its dis-PS1 mice. tance from its origin (Sholl analysis), dividing the length of the dendritic segment by the number of spines in each 10 lm stretch from the origin. DETAILED METHODS Head volume measurementIntracellular Injections With Alexa 594 Intensity thresholds were applied to each dendritic segment Mice were anesthetized with pentobarbital (0.04 mg/kg) to generate a model of the data that was visualized as a solidand transcardially perfused with 20 ml phosphate buffer (PB) surface (the Spot module, Imaris). With this module, volumefollowed by 100 ml of 4% paraformaldehyde (pH 7.4) based measurements are added to the volume rendering. Theprepared in the same buffer. The brains were postﬁxed in the Spots module models point-like structures in the data (e.g.,same solution for 24 h, and coronal sections (150 lm) dendritic spines) providing a procedure to automatically detectwere obtained on a vibratome and labeled with 10–5 M 4,6- such structures, an editor to manually correct any errorsdiamidino-2-phenylindole (DAPI, Sigma D9542). Pyramidal detected, a viewer to visualize the point-like structures asneurons in CA1 were injected individually with Alexa594 spheres, and a statistics output that includes the volume of the(Invitrogen, Eugene, OR) by passing a steady hyper- spine heads (Supporting Information Fig. 1b). A solid surfacepolarizing current through the electrode (0.5 to 21.0 nA, that exactly matched the contours of the head was created forFigs. 1a,b). The current was applied until the distal tips of each dendritic spine (Fig. 1i). The image of each dendrite waseach neuron ﬂuoresced brightly. then rotated in three dimensions and examined to ensure that the solid surface created for each spine head was correct. Head- less spines were extremely rare and they were not included in the analysis. Having taken the measurements, the 2-channelsMorphology stacks were opened to view the plaques and the spines wereConfocal microscopy categorized according to their location (within or outside of plaques). As described previously in an electron microscopy For each pyramidal neuron (5–7 neurons from each mouse, study of hippocampal dendritic spines (Trommald and Hulle-6–7 mice per group), 1–5 randomly selected dendrites were berg, 1997), we did not observe a multimodal distribution forscanned from the soma (basal dendrites) or apical trunk (apical neck length or for head volume (Fig. 1–2). Consequently, wedendrites) to the tip (125 dendrites total). In APP/PS1 mice, were unable to identify spine groups such as thin, stubby ordendrites located within the plaques were also scanned. For mushroom (Harris et al., 1992). Therefore, we chose toeach stack, the laser intensity and detector sensitivity were set describe spine morphology using measured dimensions ratherso that the ﬂuorescence signal from the spines occupied the full than shape categories.dynamic range of the detector. Therefore, while scanning, somepixels were saturated in the dendritic shaft but no pixels Neck length measurementwere saturated in the spines. In stacks containing images of Abplaques (green) the green channel was deleted. The stacks were To measure the neck length, each dendrite was visualizedcoded and the codes were not broken until the quantitative with the Volume mode of Imaris. Individual spine necks wereanalysis had been completed. measured manually in three dimensions from the interface ofHippocampus

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ALTERED SPINES IN APP/PS1 MICE 7the spine neck with the dendritic stalk to the beginning of the To estimate the plaque volume after each amyloid plaquespine head (MeasurementPro module) while rotating the was marked, the edges the plaque were marked with the Nucle-dendritic image (Knafo et al., 2009a). If the spine head was ator probe (Moller et al., 1990). The number of labeledconnected directly to the dendritic stalk, the neck length was plaques was estimated using the optical fractionator method inrecorded as zero. As the resolution in the z axis is signiﬁcantly Stereo Investigator. Plaques were marked only if their edges laylower than in the x-y axes, spines protruding perpendicular to within the dissector area and they did not intersect forbiddenthe dendritic stalk in the z axis were not measured. Thus, lines, and if they came into the focus as the optical plane60% of spines were measured. moved through the height of the dissector (20 lm). The guard zone thickness was set to 2 lm. The sum areas of the sampling site represented around 15% of the total area of the slice. ThisEstimation of the Volume and sampling method and the section interval was tested in a pilotNumber of Plaques experiment to ensure that the estimation of the number ofImmunocytochemistry plaques was representative of the total number. In slices taken from brains of the same mice used for themorphological studies, amyloid plaques were stained with an Acknowledgmentsantihuman Ab antibody in systematically sampled coronal ´ ´ The authors thank C. Hernandez and B. Garcıa for assis-sections (50 lm thick, every sixth section in the hippocampus). tance with the confocal microscopy, and Dr I. Ferrer (InstitutA pilot study conﬁrmed that this antibody exclusively marks ` Neuropatologia, Servei Anatomia Patologica, IDIBELL-Hospi-thioﬂavin-s positive plaques (Knafo et al., 2009a). Free-ﬂoating tal Universitari de Bellvitge, Universitat de Barcelona, Hospi-sections were pretreated with 55% formic acid (r-Aldrich, talet de Llobregat, Spain) for supplying the animals.ACS) and washed with 0.1 M PB. Sections were treated with1% H202 for 30 min to deplete the endogenous peroxidaseactivity, and they were then submerged for 1 h in PB with0.25% Triton-X and 3% horse serum (Vector laboratories Inc., REFERENCESBurlingame, CA). Sections were incubated overnight at 48Cwith a mouse antihuman Ab antibody (1:50; clone 6F/3D, Andersen P, Morris RG, Amaral D, Bliss T, O’Keefe J. 2006. TheDako Glostrup, Denmark) and on the following day, the Hippocampus Book. USA: Oxford University Press.sections were rinsed and incubated for 2 h with a biotinylated Bloodgood BL, Sabatini BL. 2005. Neuronal activity regulates diffu- sion across the neck of dendritic spines. Science 310:866–869.horse antimouse antibody (1:200; BA-1,000; Vector). 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